The present invention relates to a novel human gene encoding a polypeptide which is a member of the Ependymin family. More specifically, isolated nucleic acid molecules are provided encoding a human polypeptide named Ependymin. Ependymin polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting disorders related to the nervous system, and therapeutic methods for treating such disorders.
The invention further relates to screening methods for identifying agonists and antagonists of Ependymin activity.
Within the last several years, a number of ependymin have been molecularly cloned from a variety of teleost fish including Oncorhynchus mykiss (rainbow trout; Muller-Schmid, A., et al., Gene 118:189-196 (1992)), Salmo salar (Atlantic salmon; Muller-Schmid, A., et al., Gene 118:189-196 (1992)), Esox lucius (pike; Muller-Schmid, A., et al., J. Molec. Evol. 36:578-585 (1993)), Carassius auratus (goldfish; Konigstorfer, A., et al., J. Neurochem. 52:310-312 (1989); Konigstorfer, A., et al., J. Biol. Chem. 264:13689-13692 (1989)), Brachydanio rerio (zebrafish; Sterrer, S., et al., Neurosci. 37:277-284 (1990)), and Clupea harengus (herring, Muller-Schmid, et al., J. Molec. Evol. 36:578-585 (1993)). The ependymins produced by these organisms are synthesized as precursors which contain N-terminal, hydrophobic signal sequences. Each of these molecules contains multiple N-linked glycosylation sites, only some of which are conserved between species (Schmidt, R. and Shashoua, V. E. J. Neurochem. 36:1368-1377 (1981); Schmidt, R. and Shashoua, V. E. J. Neurochem. 40:652-660 (1983); Ganb, B. and Hoffman, W. Eur. J. Biochem. 217:275-280 (1993)). The precursor ependymins range in apparent molecular mass from 23.7 to 24.5 kDa, while the secreted mature forms of these molecules are typically 21.6-22.3 kDa in size.
The piscine ependymins characterized thus far may be categorized according to the number of cysteine residues present in the mature polypeptide. Mature salmoniform (O. mykiss, S. salar, and E. lucius) ependymin polypeptides contain only four cysteine residues, whereas mature cypriniform (C. auratus and B. rerio) and clupeiform (C. harengus) ependymin polypeptides contain five and six cysteine residues, respectively (Hoffmann, W. Int. J. Biochem. 26:607-619 (1994)). Correspondingly, disulfide-linked dimerization of the salmoniform ependymin polypeptides is not observed after non-reducing SDS-PAGE. However, cypriniform and clupeiform ependymins are observed as disulfide-linked dimers under non-reducing conditions. It is speculated that the dimerization occurs via the cysteine residue conserved only between the salmoniform ependymins (this cysteine residue aligns with the lysine residue at location 133 of human ependymin of the present invention as shown in SEQ ID NO:2).
Several lines of evidence have provided the basis for an understanding of the functional role(s) of the ependymins. Ca2+-binding has been demonstrated for at least goldfish and rainbow trout ependymins (Schmidt, R. and Makiola, E. Neuro. Chem. (Life Sci. Adv.) 10:161-171 (1991); Ganb, B. and Hoffman, supra). Further, ependymins are the primary cerebrospinal fluid component in a number of teleost fish (Schmidt, R. and Lapp, H. Neurochem. Int. 10:383-390 1987; ). Finally, roughly two-thirds of Ca2+ in the CSF of rainbow trout is protein-bound (Ganb, B. and Hoffman, supra). As a result, it is thought that ependymins may function in Ca2+ homeostasis of the teleost piscine brain (Hoffman, W., supra).
In situ hybridization analyses have shown that ependymins are apparently synthesized exclusively in miningeal fibroblasts of the mininx (also termed the endomeninx or leptomeninx) of teleost fish (Konigstorfer, A., et al., Cell Tissue Res. 261:59-64 (1990)). Ependymins have also been found to associate with collgen fibrils of the extracellular matrix (ECM; Schwarz, H., et al. Cell Tissue Res. 273:417-425 (1993)), and, further, have the capacity to serve as a substrate for outgrowing retinal axons (Schmidt, J. T., et al., J. Neurobiol. 22:40-54 (1991)).
An additional role for ependymins has been identified in the field of learning and memory. Using an experimental approach in which goldfish learn to swim to a specific compartment of its environment to avoid an electric shock, investigators have determined that the amount of unbound or unincorporated extracellular ependymins decreases after learning (Piront, M. -L., and Schmidt, R. Brain Res. 442:53-62 (1988); Schmidt, R. J. Neurochem. 48:1870-1878 (1987)). Further, blockage of functional ependymin molecules, either with antibodies or antisense polynucleotides, resulted in the reversible inability of the experimental animal to remember the task which it had learned. Removal of the inhibitory substance then resulted in a reappearance of the learned ability (Schmidt, R. J. supra; Shashoua, V. E. and Moore, M. E. Brain Res. 148:441-449 (1978)).
Thus, there is a need for polypeptides that function as neurotrophic factors in the regeneration of the optic and other nerves and in long-term memory consolidation, since disturbances of such regulation may be involved in disorders relating to the complex molecular and cellular process regulating neuronal and nervous system function. Such disorders may include Parkinson""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis, pain, stroke, depression, anxiety, epilepsy, and other neurological and psychiatric disorders. Therefore, there is a need for identification and characterization of such human polypeptides which can play a role in detecting, preventing, ameliorating or correcting such disorders.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding at least a portion of the Ependymin polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 or the complete amino acid sequence encoded by the cDNA clone deposited as plasmid DNA as ATCC Deposit Number 209464 on Nov. 14, 1997. The nucleotide sequence determined by sequencing the deposited Ependymin clone, which is shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1), contains an open reading frame encoding a complete polypeptide of 224 amino acid residues, including an initiation codon encoding an N-terminal methionine at nucleotide positions 296-298, and a predicted molecular weight of about 25.4 kDa. Nucleic acid molecules of the invention include those encoding the complete amino acid sequence excepting the N-terminal methionine shown in SEQ ID NO:2, or the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone in ATCC Deposit Number 209464, which molecules also can encode additional amino acids fused to the N-terminus of the Ependymin amino acid sequence.
The encoded polypeptide has a predicted leader sequence of 37 amino acids underlined in FIGS. 1A, 1B, and 1C; and the amino acid sequence of the predicted mature Ependymin protein is also shown in FIGS. 1A, 1B, and 1C, as amino acid residues 38-224 and as residues 1-187 in SEQ ID NO:2.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the Ependymin polypeptide having the complete amino acid sequence in SEQ ID NO:2 (i.e., positions xe2x88x9237 to 187 of SEQ ID NO:2); (b) a nucleotide sequence encoding the Ependymin polypeptide having the complete amino acid sequence in SEQ ID NO:2 excepting the N-terminal methionine (i.e., positions xe2x88x9236 to 187 of SEQ ID NO:2); (c) a nucleotide sequence encoding the predicted mature Ependymin polypeptide having the amino acid sequence at positions 1 to 187 in SEQ ID NO:2; (d) a nucleotide sequence encoding the Ependymin polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209464; (e) a nucleotide sequence encoding the Ependymin polypeptide having the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone contained in ATCC Deposit No. 209464; (f) a nucleotide sequence encoding the mature Ependymin polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209464; and (g) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e) or (f), above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f) or (g), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), (f) or (g), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a Ependymin polypeptide having an amino acid sequence in (a), (b), (c), (d), (e) or (f), above. A further nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of a Ependymin polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a polynucleotide which encodes the amino acid sequence of a Ependymin polypeptide to have an amino acid sequence which contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of Ependymin polypeptides or peptides by recombinant techniques.
The invention further provides an isolated Ependymin polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the full-length Ependymin polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 (i.e., positions xe2x88x9237 to 187 of SEQ ID NO:2); (b) the amino acid sequence of the full-length Ependymin polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 excepting the N-terminal methionine (i.e., positions xe2x88x9236 to 187 of SEQ ID NO:2); (c) the amino acid sequence of the predicted mature Ependymin polypeptide having the amino acid sequence at positions 1 to 187 in SEQ ID NO:2; (d) the complete amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 209464; (e) the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone contained in the ATCC Deposit No. 209464; and (f) the complete amino acid sequence of the predicted mature Ependymin polypeptide encoded by the cDNA clone contained in the ATCC Deposit No. 209464. The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those described in (a), (b), (c), (d), (e) or (f), above, as well as polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity, to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which comprises the amino acid sequence of an epitope-bearing portion of a Ependymin polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e) or (f), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Ependymin polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention.
A further embodiment of the invention relates to a peptide or polypeptide which comprises the amino acid sequence of a Ependymin polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of a Ependymin polypeptide, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
In another embodiment, the invention provides an isolated antibody that binds specifically to a Ependymin polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e) or (f) above. The invention further provides methods for isolating antibodies that bind specifically to a Ependymin polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.
The invention also provides for pharmaceutical compositions comprising Ependymin polypeptides, particularly human Ependymin polypeptides, which may be employed, for instance, to treat Parkinson""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis, pain, stroke, depression, anxiety, epilepsy, and other neurological and psychiatric disorders. Methods of treating individuals in need of Ependymin polypeptides are also provided.
The invention further provides compositions comprising a Ependymin polynucleotide or an Ependymin polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise a Ependymin polynucleotide for expression of a Ependymin polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of a Ependymin
In another aspect, a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on Ependymin binding to a receptor. In particular, the method involves contacting the receptor with a Ependymin polypeptide and a candidate compound and determining whether Ependymin polypeptide binding to the receptor is increased or decreased due to the presence of the candidate compound. In this assay, an increase in binding of Ependymin over the standard binding indicates that the candidate compound is an agonist of Ependymin binding activity and a decrease in Ependymin binding compared to the standard indicates that the compound is an antagonist of Ependymin binding activity.
In yet another aspect, the Ependymin may bind to a cell surface protein which also function as a viral receptor or coreceptor. Thus, Ependymin, or agonists or antagonists thereof, may be used to regulate viral infectivity at the level of viral binding or interaction with the Ependymin receptor or coreceptor or during the process of viral internalization or entry into the cell.
It has been discovered that Ependymin is expressed not only in primary dendritic cells, but also in the KMH2 cell line, placenta, fetal and adult liver, spinal cord, osteoclastoma, cerebellum, synovial fibroblasts, 12 week old early stage human embryo, adrenal gland tumor, whole brain, Hodgkin""s Lymphoma tissue, macrophages, HEL cell line, and chondrosarcoma. Therefore, nucleic acids of the invention are useful as hybridization probes for differential identification of the tissue(s) or cell type(s) present in a biological sample. Similarly, polypeptides and antibodies directed to those polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s). In addition, for a number of disorders of the above tissues or cells, particularly of the nervous system, significantly higher or lower levels of Ependymin gene expression may be detected in certain tissues (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a xe2x80x9cstandardxe2x80x9d Ependymin gene expression level, i.e., the Ependymin expression level in healthy tissue from an individual not having the nervous system disorder. Thus, the invention provides a diagnostic method useful during diagnosis of such a disorder, which involves: (a) assaying Ependymin gene expression level in cells or body fluid of an individual; (b) comparing the Ependymin gene expression level with a standard Ependymin gene expression level, whereby an increase or decrease in the assayed Ependymin gene expression level compared to the standard expression level is indicative of disorder in the nervous system.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of Ependymin activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated Ependymin polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of Ependymin activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an Ependymin antagonist. Preferred antagonists for use in the present invention are Ependymin-specific antibodies.